Beta carbonic anhydrases (beta-CAs) are bicarbonate-utilizing enzymes that are essential for optimal growth in many bacteria and yeast. Many bacterial beta-CAs have a unique non-catalytic bicarbonate binding site that is thought to regulate the enzyme via a conformational change that requires coordinated communication between individual protein chains in a multi-chain protein complex. The molecular basis of such inter-chain (allosteric) communication is not well-understood for this and many other allosteric enzymes, despite the fact that allosteric enzymes are both common and important in the regulation of metabolism in all organisms. This research project will use methods of molecular biology (site-directed mutagenesis), biophysical chemistry (enzyme kinetics and visible spectroscopy) and structural biology (X-ray diffraction) to probe the molecular details of intersubunit communication in beta-CA and critically test hypotheses of potential mechanisms of action. Knowledge of the molecular mechanism of intersubunit communication in beta-CA will not only provide insight into this particular (and suggest possible means of interrupting its proper function in host bacteria) but will also provide fundamental knowledge for application to mechanisms of intersubunit communication for allosteric enzymes.
Broader impacts There are several broader impacts of the research project, one of which is the involvement of undergraduate students in research projects that provide training in advanced, current praxis in protein chemistry and structural biology. A high percentage of these students, trained in the PI's lab, enter into advanced degree programs in the physical and life sciences, and ultimately join the domestic scientific work force. In addition, a post-doctoral fellow with an interest in instruction in undergraduate research and education will be trained in biophysical chemistry and structural biology. Written laboratory guides appropriate for involving undergraduates or other novice investigators in modern methodologies of protein engineering and X-ray crystallography will be updated and freely disseminated to the scientific community using a publicly accessible wiki. Finally, the acquisition of critical laboratory automation will significantly enhance the efficiency and productivity of the biophysical chemistry research infrastructure at Colgate University.
